A substrate-induced change in the stereospecificity of the serine-hydroxymethyltransferase-catalysed exchange of the alpha-protons of amino acids--evidence for a second catalytic site.

NMR has been used to study the catalysis of the hydrogen-deuterium exchange of the alpha-protons of amino acids by serine hydroxymethyltransferase (EC 2.1.2.1) from Escherichia coli. 13C-NMR was used to follow the exchange of the alpha-protons of [2-13C]glycine. The enzyme-catalysed first-order exchange rate of the pro-2S proton of glycine was approximately 7000 times more efficient than that of the pro-2R proton of glycine at both pH 7.0 and 7.8. 1H-NMR was used to follow the hydrogen-deuterium exchange rates of the alpha-protons of L- and D-2-amino derivatives of butyric, pentanoic and hexanoic acids at pH 7.8. Increasing the size of the R-group leads to a progressive change in the stereospecificity of the exchange reaction from the pro-2S proton of glycine to the 2R proton of L-amino acids. The stereospecificity for the alpha-protons of L-amino acids increased as the size of the R-group increased. With glycine, removal of tetrahydrofolate led to a large decrease in the stereospecificity of the exchange reaction but did not affect the exchange rates of the alpha-protons of any of the larger amino acids studied. We show that the Schiff base formed between L-2-aminohexanoic acid (L-norleucine) and pyridoxal 5'-phosphate binds at a different site from the Schiff base between glycine and pyridoxal 5'-phosphate. The molecular basis of these results is discussed.